A variety of biological sensors which utilize fluorescence in the detection of molecules are known in the art. Sensors in which molecular recognition coupled with fluorescent quenching for example, are used for the detection of analyte concentrations in a variety of bioassays. In addition to their in vitro applications, the use of biosensors in in vivo is of particular interest to the medical community due to their potential in the amelioration a variety of disease conditions. For example, the concept of implantable biological sensors which can continuously measure glucose levels in diabetic individuals has existed for several decades. A primary goal in this art has been to overcome the disadvantages of capillary blood glucose self-monitoring by developing a glucose sensor, which can provide more frequent and easily acquired glucose information. In addition, an ideal sensor can function as a hypoglycemic and hyperglycemic alarm, and ultimately serve as the controller for an artificial endocrine pancreas. In this context, non-invasive glucose sensors are of particular interest to medical practitioners, and typical approaches to non-invasive blood glucose measurement are described in U.S. Pat. Nos. 4,428,366, 4,655,225, 4,805,623, 4,875,486, 4,882,492, 5,028,787, 5,054,487, 5,070,874, 5,077,476, 5,086,229, and 5,112,124, the disclosures of each being incorporated herein by reference.
The continuous, long-term, real-time ambulatory measurement of glucose concentrations in vivo is an important problem that remains to be solved. The in vitro equivalent is the ability to sense glucose continuously under aqueous physiologically relevant conditions, a challenging enough proposition due to restrictions placed on pH, buffer and temperature. The long-term, continuous glucose measurement problem is of particular significance in diabetes, where such measurement is of crucial importance to ensure proper glycemic control in insulin dependent individuals, and to determine glucose levels for any reason even in non-insulin dependent diabetics. Such measurement in vivo, if capable of being performed in a minimally invasive or non-invasive fashion, would be of tremendous significance, and contribute greatly to the lives of at least 16 million afflicted people in the U.S. alone.
Measurements of glucose might be performed by direct spectroscopic signatures of the glucose molecule, or via the aid of chemical or biological receptors for the glucose molecule, wherein a binding or a binding related event is coupled to a signal transducing method so as to be read either by spectroscopic, amperometric or related means. With a suitable calibration method, such data would enable a continuous glucose measurement. Of the chemical receptors, one of particular note is any boronate containing species since boronates reversibly bind polyols, glucose being one good example of such species. James et. al. describe a phenylboronic acid moiety coupled with a fluorescent molecular component as organic compounds that act as chemical biosensors for polyhydroxylated molecules (see, e.g. James, T. D., et. al., J. Chem. Soc. Chem. Commun., 1994, 477-478). The sensing capabilities of these molecules are determined by the changes (increases or decreases) in fluorescence intensity exhibited upon binding of a polyhydroxylated saccharide, of which glucose is of particular interest. The binding event may be recorded by any proximal reporter species that is capable of sensing and signaling this binding. Colorimetric methods, in which a change is induced by direct or indirect chemical or physical commerce between the boronate and the actual portion reporting the change is fairly common. Fluorescence reporters are of particular value because of the low levels of analyte capable of being sensed, the low amounts of fluorophore required, the inherent sensitivity of fluorescence, and the two or three well-established methods of detecting fluorescence phenomena.
When combined with existing insulin pump technologies, a minimally-invasive, continuous glucose sensor is of great benefit to patients in achieving tighter blood-glucose control. The incorporation of phenylboronic fluorescent compounds in a sensor designed to detect glucose is described in U.S. Pat. No. 6,002,954 to Van Antwerp et al., which discloses an implantable optical sensor designed to facilitate the management of diabetes. In this sensor system, a fluorescent transducer is implanted 1-3 mm below the surface of the skin and optically interrogated externally to determine the level of tissue glucose in diabetic patients.
A number of biological sensors known in the art utilize functional moieties incorporated into macromolecular matrices (e.g. polymers). In this context, the use of such matrices in biological sensors can provide a number of advantages in sensor design, manufacture and use including ease in manipulation. While molecular glucose sensing species have been synthesized and tested, there is a need for in vivo sensors which incorporate these species. In addition, while certain polymer-based sensors are known in the art, there is a need for improved macromolecular matrices which can attach glucose sensing species in an active sensor and methods for making such matrices. Specifically, there is a need in the art for macromolecular matrices which can be manipulated to incorporate molecularly tailored polyhydroxylate sensing species and related calibration moieties in order to produce sensors having optimized characteristics. Embodiments of present invention fulfill these needs and provide other related advantages.